林中天臺灣大學:獸醫學研究所林念儀Lin, Nien-YiNien-YiLin2010-05-042018-07-092010-05-042018-07-092009U0001-2007200911421100http://ntur.lib.ntu.edu.tw//handle/246246/178944脂肪組織為身體的重要器官，掌管體內能量恆定，且可釋放多種激素以調節體內各種生理功能，脂肪細胞分化的異常可能引起肥胖以及相關的疾病。在以3T3-L1脂肪前驅細胞為模式的研究中發現，當細胞受到分化刺激時，多種極早期基因(immediate early gene)的表現會受到活化，包括RNA降解因子tristetraprolin (TTP)，TTP可藉由與mRNA 3’端非轉譯區(3’ untranslated region, 3’UTR)中特殊的多腺嘌呤-尿嘧啶序列(AU-rich element, ARE)結合而加速其降解，本論文的目標即在瞭解TTP在調節脂肪細胞分化過程中的角色。將與TTP結合的mRNA分離並鑑定後，發現TTP的表現可受到自我調控，TTP可藉由與自己的mRNA結合而降低本身的表現。本研究也分離到TTP的新標的─MKP-1 mRNA。研究發現當脂肪細胞受到分化刺激後，ERK pathway同時活化TTP與MKP-1的表現，MKP-1的表現可抑制ERK活性，進而造成此一訊息傳遞的負調控。而MKP-1的表現則受到TTP及另一個RNA結合因子HuR的調控，TTP與MKP-1 mRNA的結合造成RNA降解而抑制MKP-1表現，HuR與MKP-1的結合則可使MKP-1 mRNA更加穩定。MKP-1 3’UTR具有三段ARE，在RNA IP、RNA pull-down及REMSA等實驗中證實他們對TTP及HuR有不同的親和力，證據指出TTP與HuR皆傾向與ARE1和ARE2結合，對ARE3的結合力較低，有趣的是，即便如此，TTP仍可藉由其他不明方式調節ARE3。進一步研究發現，TTP對RNA的親和力可受到轉譯後修飾機制(posttranslational modification)的調節。p38 pathway的活化使TTP呈高度磷酸化的狀態，且對RNA的親和力降低；而ERK pathway造成的磷酸化雖然對TTP的分子量影響不大，但卻能有效增加其對RNA的親和力。另外，在進行RNA干擾(RNAi)實驗後發現，抑制TTP的表現同時也抑制脂肪細胞分化，但抑制MKP-1表現後，脂肪細胞的複製擴增(mitotic clonal expansion)增加且分化的程度更加提高，此結果顯示TTP及MKP-1在調節脂肪細胞分化有重要的功能。綜合上述，本研究顯示MAPK pathway可活化TTP基因的轉錄，又可藉由轉譯後修飾機制影響TTP的功能，而TTP可藉由調節MKP-1的表現而影響MAPK pathway的活性，彼此間形成精密的調控網絡，進而控制脂肪細胞分化。Dysregulation of adipogenesis has been considered as a cause of the development of obesity and obesity-related diseases. Among the immediate early genes (IEGs) activated during the differentiation of 3T3-L1 preadipocytes, we identified tristetraprolin (TTP), a zinc finger-containing RNA-binding protein which was reported to bind AU-rich elements (AREs) of target mRNAs and induce their rapid degradation. To understand how TTP may be involved in regulating adipogenesis, the expression and mRNA targets of TTP were analyzed. Biochemical and functional assays showed that the expression of TTP per se was autoregulated. Sequence analysis of 3’UTR of IEG mRNAs differentially bound by TTP suggested a preferential binding sequence of UUAUUUAUU. In addition to TTP and the previously reported COX-2 mRNAs, we have also identified MKP-1 mRNA as a novel target of TTP. The expression of MKP-1 and TTP was simultaneously activated by ERK signaling, while MKP-1 protein functions as a feedback regulator of ERK. Moreover, the expression of MKP-1 was tightly controlled by ARE-binding proteins TTP and HuR. Binding of TTP to MKP-1 mRNA resulted in rapid decay and binding of HuR resulted in mRNA stabilization. The three AREs in MKP-1 3’UTR were differentially bound by TTP and HuR. More specifically, both of TTP and HuR preferentially bound to MKP-1 ARE1 and ARE2, but showed low binding affinity to MKP-1 ARE3. The RNA-binding affinity of TTP was posttranslationally modified by p38 and ERK signaling. TTP exhibited a decreased RNA binding while being phosphorylated by p38 signaling and showed an enhanced RNA binding while being phosphorylated by ERK signaling. The functional importance of TTP and MKP-1 during 3T3-L1 differentiation was further demonstrated by siRNA knockdown experiment. These results suggested that TTP can regulate the MKP-1 mRNA stability to subsequently control the activation of MAPK signaling pathways in adipocyte differentiation.誌謝 ................................................................................................................... i文摘要 ....................................................................................................................... iiibstract ......................................................................................................................... vontents ......................................................................................................................... viibbreviations ............................................................................................................... xi. Introduction .................................................................................................................. 11. Adipogenesis and diseases .................................................................................. 12. Molecular regulation of adipogenesis ................................................................ 10. Modulation of gene expression through posttranscriptional regulation ............ 234. TTP-regulated RNA decay ............................................................................... 33I. Material and Methods .............................................................................................. 43. Cell culture .........................................................................................................43. Plasmid constructs ............................................................................................. 433. RNA extraction and reverse-transcription ....................................................... 45. Semiquantitative PCR ....................................................................................... 455. Real-time PCR ……………….......................................................................... 46. Preparation of whole cell extracts and cytoplasmic/nuclear extracts and estern blotting assay ..................................................................................... 47. RNA pull-down assay ........................................................................................ 48. RNA-immunoprecipitation assays ................................................................... 49. RNA EMSA ...................................................................................................... 500. Luciferase reporter assay and Northern blotting ........................................... 511. RNA interferences .......................................................................................... 52II. Specific Aims ......................................................................................................... 53V. Results ................................................................................................................... 55. Analysis of expression kinetics of TTP and the possible autoregulation uring 3T3-L1 differentiation .......................................................................... 55. Identification of novel TTP-targeted mRNAs during 3T3-L1 differentiation … 60. Molecular functions of MKP-1 during 3T3-L1 differentiation ……………….. 64. ERK signaling modulates MKP-1 mRNA expression transcriptionally and osttranscriptionally .......................................................................................... 65. Physical interaction between TTP and three MKP-1AREs …………………… 67. HuR stabilized MKP-1 mRNA through interaction to MKP-1 ARE1 andRE2 ................................................................................................................ 70. Posttranslational modification regulated the RNA-binding affinity of TTP ….. 70. Knocked-down MKP-1 expression enhanced adipocyte differentiation ……… 72. Conclusions ............................................................................................................. 75I. Discussion ............................................................................................................... 77II. Figures .................................................................................................................. 85igure 1 Morphological changes of 3T3-L1 during differentiation ………………… 86igure 2 Expression of TTP mRNA and protein during differentiation of 3T3-L1readipocytes .............................................................................................. 87igure 3 Expression of TTP mRNA and protein stimulated by different inducers … 89igure 4 Half-life of TTP mRNAs shortened after induction to differentiate ….…… 91igure 5 Interaction between TTP and HuR proteins and TTP AREs ….…………… 92igure 6 Functional characterization of TTP and HuR-mediated ARE-containing TP mRNA expression .............................................................................. 97igure 7 RNA interferences of TTP expression down-regulated 3T3-L1 ifferentiation .............................................................................................. 100igure 8 mRNA metabolism of the IEGs during the early differentiation of 3T3-L1cells ........................................................................................................... 102 igure 9 TTP could interact with MKP-1 and COX-2 mRNA. (A) RNA-IPnalysis .................................................................................................... 103igure 10 Expression profiling of MAPKs and MKP-1 during differentiation ofT3-L1 cells ….......................................................................................... 105igure 11 ERK signaling regulates MKP-1 mRNA expression in differentiation f 3T3-L1 cells .................................................................................. 108igure 12 ERK signaling induced TTP expression and downregulated MKP-1 RNA stability ...................................................................................... 109igure 13 Interaction between MKP-1 AREs and endogenous TTP and HuR rom 3T3-L1 cytosol ............................................................................. 111igure 14 MKP-1 AREs mediated posttranscriptional gene regulation and wereown-regulated by TTP overexpression ................................................. 113igure 15 TTP showed strong interaction to MKP-1 ARE1 and ARE2, but weaknteraction to ARE3 ………….………………………………………….. 116igure 16 HuR stabilized MKP-1 mRNA stability by interacting with ARE1 and RE2, but not with ARE3 ........................................................................ 119igure 17 Posttranslational modification regulated the RNA-binding affinitynd RNA-destabilizing effect of TTP .................................................. 121igure 18 Inhibited TTP and MKP-1 expression altered adipocyte differentiation rocesses ................................................................................................... 124igure 19 Hypothesized regulatory networks between MAPKs, MKP-1 andNA-destabilizing factor TTP during the early differentiation ofT3-L1 cells. …………………………………………………………..... 125III. Tables .................................................................................................................. 127able 1 Primers used to characterize the IEGs expression in differentiation of T3-L1 cells by real-time PCR ................................................................... 127able 2 Characteristics of IEG’s AREs ..................................................................... 128X. Supplemental Data ................................................................................................ 129igure 1 HuR could interact with ARE-containing IEG mRNAs ............................. 129. References ............................................................................................................... 131application/pdf9503142 bytesapplication/pdfen-US脂肪細胞分化多腺嘌呤-尿嘧啶序列TTPMKP-1轉錄後調節機制轉譯後修飾機制adipogenesisAREposttranscriptional regulationposttranslational modification[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178944/1/ntu-98-F90629016-1.pdf